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Multiple Toxin Production in the Cyanobacterium Microcystis: Isolation of the Toxic Protease Inhibitor Cyanopeptolin 1020

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Multiple Toxin Production in the Cyanobacterium Microcystis: Isolation of the Toxic Protease Inhibitor Cyanopeptolin 1020 Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural Products, 73(5):980-984. Postprint available at: http://www.zora.uzh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch Originally published at: Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the Winterthurerstr. 190 cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural CH-8057 Zurich Products, 73(5):980-984. http://www.zora.uzh.ch Year: 2010 Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020 Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural Products, 73(5):980-984. Postprint available at: http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch Originally published at: Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural Products, 73(5):980-984. Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020 Abstract The isolation and structure of cyanopeptolin 1020 (hexanoic acid-Glu-N[-O-Thr-Arg-Ahp-Phe-N-Me-Tyr-Val-]) from a Microcystis strain is reported. Very potent picomolar trypsin inhibition (IC(50) = 670 pM) and low nanomolar values against human kallikrein (4.5 nM) and factor XIa (3.9 nM) have been determined for cyanopeptolin 1020. For plasmin and chymotrypsin, low micromolar concentrations were necessary for 50% inhibition. Cyanopeptolin 1020 was found to be toxic against the freshwater crustacean Thamnocephalus platyurus (LC(50) = 8.8 microM), which is in the same range as some of the well-known microcystins. These data support the hypothesis that cyanopeptolins can be considered as a second class of toxins in addition to the well-established microcystins in Microcystis. 1 Multiple Toxin Production in the Cyanobacterium Microcystis: Isolation of the Toxic Protease Inhibitor Cyanopeptolin 1020 Karl Gademann†,‡,*, Cyril Portmann, ‡ Judith F. Blom,§ Michael Zeder, § and Friedrich Jüttner§ Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel, Switzerland Chemical Synthesis Laboratory, SB-ISIC-LSYNC, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland Limnological Station, Institute of Plant Biology, University of Zürich, CH-8802 Kilchberg, Switzerland * To whom correspondence should be addressed. Tel: ++41 61 267 11 44. Fax: ++41 61 267 11 45. E-mail: [email protected] † University of Basel ‡ Swiss Federal Institute of Technology § University of Zürich 2 The isolation and structure of cyanopeptolin 1020 (hexanoic acid-Glu-N[–O–Thr- Arg-Ahp-Phe-N-Me-Tyr-Val-]) from a Microcystis strain is reported. Very potent picomolar trypsin inhibition (IC50 = 670 pM) and low nanomolar values against human kallikrein (4.5 nM) and factor XIa (3.9 nM) have been determined for cyanopeptolin 1020. For plasmin and chymotrypsin, low micromolar concentrations were necessary for 50% inhibition. Cyanopeptolin 1020 was found to be toxic against the freshwater crustacean Thamnocephalus platyrus (LC50 = 8.8 µM), which is in the same range as some of the well-known microcystins. These data support the hypothesis that cyanopeptolins can be considered as a second class of toxins in addition to the well-established microcystins in Microcystis. 3 Cyclic depsipeptides containing the structural element 3-amino-6-hydroxy-2- piperidone (AHP) (so-called cyanopeptolins) are widely distributed secondary metabolites in cyanobacteria.1 The structural diversity of previously described compounds is large, but all can be considered inhibitors of serine proteases of crustaceans and mammals.1 Although preferentially mammalian enzymes in the context of pharmacological research have been tested, the evolutionary primary target enzymes can be assumed to be those of aquatic invertebrates that are important grazers of these cyanobacteria.1 Digestive serine proteases of these invertebrates are strongly inhibited2 and could thus render the cyanobacteria unsuitable food for them. The conserved feature of serine proteases in evolution allows these inhibitors to be successfully applied also against mammalian enzymes. Most of the AHP-containing compounds are active in the micromolar range,1c but some fit so perfectly in the reaction center of mammalian serine proteases that nanomolar concentrations were shown to be sufficient for inhibition.3 Co-crystallization experiments have been conducted to analyze in atomic detail the binding properties of such an inhibitor to the reaction center of mammalian trypsin.4 While the inhibition of digestive serine proteases of various organisms is well established, on the acute toxicity of some of these congeners is not understood. Microcystilide A when applied intraperitoneally to mice induced convulsions and spasms but not death.5 Cyanopeptolin SS isolated from a natural cyanobacterial bloom caused death of crustaceans6 as did oscillapeptin J for Thamnocephalus platyurus.7 While little is known about the toxicity of cyanopeptolins, the most studied cyanobacterial toxins are the microcystins, which exert their hepatotoxicity through protein phosphatase inhibition8 and are produced by species of the genera Microcystis, Anabaena, Oscillatoria, Planktothrix, and Nostoc. Microcystins are 4 suspected of countless animal poisonings throughout the globe and also thought to be responsible for a severe incident in Brazil, where several persons died of contaminated water.9 As a direct consequence, the World Health Organization set guidelines for drinking water and defined threshold values for microcystin concentration.9 However, cyanobacteria of the genus Microcystis are known to produce several classes of compounds in addition to the microcystins frequently totaling more than 10 distinct secondary metabolites. For example, Microcystis aeruginosa PCC 7806 is known to produce cyanopeptolins A-D, cyanopeptolin 963A, [D-Asp3]microcystin-LR and microcystin-LR, and the aerucyclamides A-D.10 While most of the attention on toxic cyanobacterial peptides focused on the microcystins, the above mentioned reports on toxic effects of cyanobacteria might imply the presence of additional toxins related to cyanopeptolins in Microcystis. Thus, one could propose that potent and unselective trypsin inhibitors are able to shut down other processes than protein digestion by virtue of their binding to other serine proteases. Here we report on an extremely potent, picomolar trypsin inhibitor, cyanopeptolin 1020 (1), which also binds in the low nanomolar range to factor XIa and human kalllikrein. In addition, this compound exhibits acute toxic properties against the crustacean Thamnocephalus platyurus. In addition to several microcystins found in the producing strain, cyanopeptolin 1020 (1) constitutes a second toxic compound class, which thus provides supporting evidence for multiple toxin production in Microcystis. 5 NH O 2 OH H2N N N N H H O NH O O H N O N CH3 O H O O N N CH3 H O O O OH Cyanopeptolin 1020 (1) CH CO2H 3 O N HN NH CH3 CH3 H3C O CH2 O O O NH HN H H H CH3 CH3 N N O CH3 O CO2H O H3C CH3 Microcystin-LA The 60% methanolic extract of the biomass of Microcystis aeruginosa UV-006 was fractionated and assayed against the sensitive freshwater crustacean Thamnocephalus platyurus for acute toxicity. Overall, more than 20 different compounds were present in these fractions, and out of totally 16 fractions, nine were found to display acute toxicity in this assay. Among these nine fractions, eight compounds were assigned as known or new microcystins (Table S1, see supporting information). So far microcystins of all Microcystis strains have been shown to contain the structural element N-methyldehydroalanine rather than the isomeric dehydrobutyrine. Under this presumption, we found the very common and typical microcystin congeners [Asp3]-MC-LR and MC-LR. MC-LR and MC-LA have already been described in a previous study on this strain11 and we could confirm both components. In addition, we 6 found the lipophilic congeners MC-LAba, MC-LA, MC-LV and MC-LL which were only rarely reported before.12 Additionally, two new variants were found (assigned based on typical absorption and mass spectra), which have not been described yet (see table S1, supporting information). However, the amounts available were too small to allow the determination of the structure. Interestingly, one compound displayed similar toxicity as MC-LA, but based on the UV spectrum, it was hypothesized that this compound is not a microcystin, but a structurally different peptide. In order to elucidate the structure of this toxin, this fraction was purified and analyzed by NMR spectroscopy and chemical degradation (amino acid analysis). The molecular formula of 1 was determined to be C50H72N10O13 based on HRMS complemented by isotope labeling studies that established the presence of 10 N atoms. This molecular formula was consistent with the NMR data (Table 1). The 1H NMR spectrum displayed the typical pattern of a peptide. The different amino acids were identified based on 1H,1H COSY, 1D- TOCSY and HMBC data as Glu, Thr, Arg, Ahp, Phe, N-Me-Tyr, Val and hexanoic acid (HA), see also the supporting information for full details. The sequence was obtained by 1H-13C long-range correlations: From Glu-NH to HA-C1, from Thr-NH to Glu-C1, from Arg-NH to Thr-C1, from Thr-H3 to Val-C1, from Val-NH to N-Me- Tyr-C1, from N-Me-Tyr-NCH3 to Phe-C1 and from Ahp-NH to Arg-C1. The macrocyclic ring was established by a ROESY interaction between Ahp-H5 and Phe- H3a, and Ahp-NH and Arg-H2 (Figure 1).
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